Spinally-projecting serotonergic neurons have been suggested to be involved in the modulation of pain and in the mediation of opiate analgesia. However, it has been unclear in what ways these neurons are organized. Recently it was observed that the serotonergic fibers and varicosities found in the most of the spinal cord also contain substance P (SP) and/or thyrotropin-releasing hormone (TRH). In contrast, the serotonergic fibers and varicosities of the superficial dorsal horn appear to contain neither SP nor TRH. The fact that the superficial dorsal horn has been suggested to be involved in the expression of pain suggests the working hypothesis of this grant: That the serotonergic neurons in which neither SP nor TRH occur constitute a specific system for the modulation of pain. This hypothesis will be examined by both anatomical and electrophysiological means. The distribution of spinally- projecting serotonergic neurons in which neither SP nor TRH occur ("5HT-only" neurons) will be mapped using a combination of 3-color immunofluorescence and fluorescent retrograde tracing. In addition, numbers of cells appearing to receive input from "5HT-only" varicosities will be compared in two systems of ascending somatosensory neurons: spinothalamic tract neurons, which are nociceptive, and post-synaptic dorsal column neurons, which appear not to be nociceptive in the rat. Finally, the distribution of "5HT-only" fibers and varicosities will be examined in the gray and white matter of the spinal cord. In all of these experiments it is expected that "5HT-only neurons will be selectively associated with nociceptive systems. In the electrophysiological experiments, putative serotonergic neurons will be antidromically activated from the superficial dorsal horn using microstimulation techniques. The characteristics of these neurons will then be examined in terms of their responses to innocuous and noxious stimuli, and to systemic morphine. It is expected that these neurons will be inhibited by noxious stimuli and excited by morphine.